The long-term objective of this study is to understand the phenotypic development of precerbellar neurons that are involved in error detection and correction of horizontal visual tracking. Zebrafish will be employed because of the strength of the available genetic tools and the ability, through optical means, to concurrently monitor behavior and neural activity in this model system. A longitudinal study quantifying horizontal eye tracking behavior will be used to determine when velocity sensitivity is acquired in precerbellar Area II and inferior olive (IO) neurons. This acquisition is a prerequisite for the onset of the visual error correction. Based on work in the adult goldfish, Area II neurons code for eye velocity and provide input to the cerebellum via mossy fibers, while the IO neurons give rise to the climbing fibers and signal retinal-slip velocity. In larval zebrafish horizontal eye movements are produced by Hox3 dependent networks arising in rhombomeres (rhs) 5-6.The precerebellar neurons necessary for optimizing horizontal behaviors through plasticity originate in rhs 7-8 under the influence of Hox4 genes. Hox4 expression is hypothesized to be necessary for the development of velocity sensitivity in Area II and the IO. Using a two-photon laser-scanning microscope, the Ca2+ dynamics of Hox4 expressing neurons marked by a fluorescent signal, YFP, will be monitored while the animal responds to a visual stimulus. These dynamics reflect firing rate activity and thus can be used to monitor the level of velocity sensitivity in active neurons. The Ca2+ dynamics of marked and unmarked neurons will be compared with regard to whether and at what age these neurons acquire velocity sensitivity. Additionally, morpholinos designed to knock down Hox4 expression will be combined with investigations of physiology and anatomy to further probe the influence of Hox4 expression on the development of velocity sensitivity. The first aim of the proposed research will be to conduct a longitudinal study of the YFP positive and negative precerebellar neuronal distributions in Area II and IO with regard to velocity sensitivity. The second aim will be to ask whether Hox4 expression plays a role in the cellular anatomy and physiology underlying velocity sensitivity. Completion of this work will set the stage for studying oculomotor learning and memory that are entirely dependent on the Area II mossy and IO climbing fiber connections to the cerebellum.